The Study Of Three Mini-CRISPR Systems In Tobacco And Cabbage And Development Of A Leaf Vein Pigment Marker Editing Systems

CRISPR/Cas,a powerful gene editing tool,is an"immune system"that has been unearthed in bacteria and archaea,whereCas nucleases,led by sgRNA,can target and cleave target sequences to obtain targeted editing products.The current mainstream gene editing tools,such as CRISPR/Cas9,have largeCas proteins,usually larger than1000 amino acids,which limit the loading of other functional components and are not conducive to their delivery and application in plant and animal cells.Thus,three newly mined MiniCas proteins,Un1Cas12f1,SpCas12f1 and AsCas12f1,which are about half the size ofCas9 volume,have been recently unearthed.The cleavage activities of the above three Mini-Cas proteins were verified in human cell genome and bacteria,and in plants only SpCas12f1 was validated for editing function in maize and rice.Therefore,this experiment utilized these threeCas12f1 nucleases as editing tools in combination with three different sgRNA expression as well as release structures:sgRNA-tRNA,sgRNA-HDV,and sgRNA-TTTTTATTTTTT,considering high temperature treatment,in tobacco for the Nt PDS gene and in cabbage for the Bo Wax gene.At the same time,previous studies in our laboratory have shown that 35S::MYB transformed plants exhibit anthocyanin accumulation in roots,stems and leaves,which affects the normal growth and development process of the plants.Therefore,in this experiment,we selected three leaf vein-specific promoters,fused MYB genes,and studied their expression patterns in tobacco in order to obtain a visual selection marker that expresses anthocyanins specifically in leaf veins,has no significant effect on the growth and development of the recipient plants,and is easy to identify.It was also fused with a gene editing system to construct a leaf vein pigment marker-based gene editing system to facilitate high-throughput screening of mutant progeny without transgenic components.The results of this pilot study are as follows:1.The Mini-CRISPR systems AsCas12f1,SpCas12f1 and Un1Cas12f1 enable editing events with limited efficiency in tobacco.In this experiment,three MiniCas proteins:Un1Cas12f1,SpCas12f1,AsCas12f1,were used to take three sgRNA expression and splice release structures:sgRNA-tRNA,sgRNA-HDV,sgRNA-T₄AT₆,considering the PAM preference of eachCas protein.Spacer sequences of 18 bp or 20 bp length were selected,and a total of 16 expression vectors were constructed and validated in tobacco with short culture cycles to test the editing function and efficiency of the Mini-CRISPR system in tobacco.Meanwhile,five normally grown guaired tissues were selected from each vector and subjected to variable temperature treatment,（45°C,4h/d）x3.Sanger sequencing analysis was performed after amplification of target sites of transgenic tobacco plants under both normothermic and variable temperature treatment,and no editing occurred at the target sites.Meanwhile,by deep sequencing analysis of Site 1 and 2 amplicons,the sgRNA-HDV structure under 25°C condition only,Un20-4-5 plants detected 0.21%T→C at Site1 and 0.22%A→G and 0.25%C→T base substitutions near Site1.In addition,in the sgRNA-T₄AT₆structure,low frequency base substitutions were detected in variable temperature treated AsCas12f1,SpCas12f1 and Un1Cas12f1samples.Among them,plant Sp20-3-1 detected 0.78%G→A at Site 1 and 0.86%A→G and 0.74%T→C near Site 1;plant Un20-4-15 detected 0.21%T→C at Site 1;and plant As18-3-7,0.39%C→T base substitution occurred near Site 2 substitution near Site 2.2.AsCas12f1 and Un1Cas12f1 based on sgRNA-tRNA structure failed to achieve gene editing function in cabbage.In this experiment,we targeted kale wax powder synthesis-related gene Bo Wax,released sgRNA-Spacer product by sgRNA-tRNA structure,selected target sites of different lengths of 18 bp or 20 bp,and constructed plant expression vector to transform kale.Sanger sequencing and Site A amplicon deep sequencing analysis of the Bo Wax target site of the transgenic plants did not detect the occurrence of editing events at the target site.3.A visual selection marker gene editing system based on anthocyanin accumulation in leaf veins was established.In this experiment,promoters were selected from Arabidopsis PSul,PAmi promoter and tomato PObe promoter fused with MYB gene to transform tobacco leaf discs using 35S::MYB as control.The results showed that the PAmi promoter could not produce anthocyanin accumulation in any tissue of tobacco seedlings,and both PSul and PObe promoters could normally drive MYB genes to cause anthocyanin accumulation in tobacco leaves,petioles,and stems,but both were higher in leaf veins.Combined with the PSul::GUS and PObe::GUS staining results,it is hypothesized that these two promoters drive MYB gene expression followed by anthocyanin accumulation in the chloroplasts as well,which may be related to anthocyanin transport through the vascular tissue.Compared with the control,both leaf vein-specific promoters were able to drive MYB expression normally without affecting the plant growth and development process,and flowering time was 3-4 mths earlier than that of the control.In addition,the three truncated promoters PSul1/PSul2/PSul3targeting PSul also efficiently accumulated anthocyanins in tobacco leaf veins.PSul3::MYB was selected as the pigment marker gene in pCA-AsCas12f1-Wax and pCACas9-Dw gene editing vectors to transform cabbage and tobacco,respectively.In cabbage transgenic plants,anthocyanin accumulation was produced mainly in stems,petioles and dorsal leaf veins.In the tobacco dwarf mutant plants,the expected bright purple color appeared on the leaf surface and obvious anthocyanin accumulation was visible on the abaxial leaf veins.The editing efficiency of the target gene reached62.96%,which was comparable to the editing efficiency of the pigment-free marker pCACas9-Dw knockout vector in our laboratory.